Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Imprinted proteins for determination of ovalbumin.

A strategy to design imprinted proteins (IPs) able to detect a glycoprotein (ovalbumin, OVA) was proposed. Glucose oxidase (GOx) was used as a matrix for obtaining the binding cavities with high specificity towards the template protein. The effective method to purify the obtained IPs from the template molecules was developed based on a combination of dialysis and gel filtration. HRP was chosen as a model template to discover the optimal production conditions, and the optimal template concentration (100 µg⋅L-1) was chosen. The obtained imprinted proteins were characterized by the high adsorption selectivity towards the target protein (the imprinting factor towards OVA and HRP is 4.7). The developed method was transferred to the synthesis of the anti-OVA IPs. The binding properties of these IPs were estimated using the OVA conjugates with low- (FITC) and high- (HRP) molecular weight label molecules. The ability of the synthesized anti-OVA IPs as recognition elements in immunoassay was studied. Under the optimized experimental conditions, the proposed imprinted proteins exhibited a good linear response to OVA in the concentration range of 10-2000 ng⋅mL-1 with a detection limit of 6 ng⋅mL-1. The obtained recognition elements were tested for OVA determination in real samples of chicken egg white, and a sample of OVA-free cake spiked by OVA.

Immunochemical approaches for detection of aflatoxin B1 in herbal medicines.

INTRODUCTION: Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary metabolite of Aspergillus flavus and A. parasiticus. AFB1 was classified as a Group I carcinogen by the World Health Organisation for Research on Cancer in 1993. AFB1 is an unavoidable natural contaminant of some herbal medicine, able to cause serious health issues for humans consuming the related medicine. OBJECTIVE: Therefore, this study aimed to develop an efficient fluorescence polarisation immunoassay (FPIA) and a rapid, low-cost, and easy-to-use membrane-based flow-through immunoassay (MBA) for determination of AFB1 in herbal medicine Origanum vulgare L., Rubus idaeus L., Urtica dioica L. and Sorbus aucuparia L. RESULTS: A cut-off level of the developed MBA was 0.8 ppb. Validation of the developed test was performed with blank and spiked samples. Using three naturally contaminated or three artificially spiked samples. The FPIA showed a linear working range of 8.6 to 64 ppb, and a half maximal inhibitory concentration (IC50 ) of 24 ppb. CONCLUSION: The results were in good correlation with the enzymelinked immunosorbent assay (ELISA) results (the IC50 0.1 ppb). Both the sample preparation and analysis are simple, cost-effective and easy to perform on-site in non-laboratory environments. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used as a confirmatory technique.

Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO2). Then we applied the QD@SiO2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500µgkg-1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result.

The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.

CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.

Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization.

Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.

Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

Design of a sensitive fluorescent polarization immunoassay for rapid screening of milk for cephalexin.

In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 µg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 µg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 µg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.

Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction.

The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3'→5' exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3'-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.

Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.

We report on the synthesis of core-shell CuInS2/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS2/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay.

Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S] cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus.

Cas4 proteins, a core protein family associated with the microbial system of adaptive immunity CRISPR, are predicted to function in the adaptation step of the CRISPR mechanism. Here we show that the Cas4 protein SSO0001 from the archaeon Sulfolobus solfataricus has metal-dependent endonuclease and 5'→3' exonuclease activities against single-stranded DNA, as well as ATP-independent DNA unwinding activity toward double-stranded DNA. The crystal structure of SSO0001 revealed a decameric toroid formed by five dimers with each protomer containing one [4Fe-4S] cluster and one Mn(2+) ion bound in the active site located inside the internal tunnel. The conserved RecB motif and four Cys residues are important for DNA binding and cleavage activities, whereas DNA unwinding depends on several residues located near the [4Fe-4S] cluster. Our results suggest that Cas4 proteins might contribute to the addition of novel CRISPR spacers through the formation of 3'-DNA overhangs and to the degradation of foreign DNA.

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

A novel electrochemical sensor for the detection of fipronil and its toxic metabolite fipronil sulfone using TiO2-polytriazine imide submicrostructured composite as an efficient electrocatalyst.

For the first time, a simple and sensitive electrochemical sensor based on a screen printed electrode (SPE) modified with titanium dioxide (TiO2) and polytriazine imide submicrostructured composite (TiO2-PTI) has been developed for the simultaneous detection of fipronil (FIP) and its toxic metabolite fipronil sulfone (FIP-S). The submicrostructured composite material based on TiO2 and PTI was obtained by simple hydrothermal treatment of the Ti peroxocomplexes in the presence of pristine. This carbon nitride allotrope has better crystallinity and conductivity than its graphitic analog. It was found that the TiO2-PTI submicrostructured composite enhanced the electrochemical sensing of the SPE electrode towards FIP and its metabolite FIP-S in 0.1 M Britton-Robinson buffer (pH 10) at the oxidation potentials of 0.82 V and 0.94 V, respectively. In addition, it showed good stability and reproducibility for the determination of both analytes. Under optimal conditions, the peak currents by square wave voltammetry were found to vary linearly with FIP and FIP-S concentrations in the range from 0.01 to 10 µM and from 10 to 50 µM, with a detection limit of 8.42 nM, 3.6 µg/kg for FIP and 9.72 nM, 4.04 µg/kg for FIP-S. This sensor was successfully used to detect FIP and FIP-S in eggs and water samples with good recoveries of 90%-106.6%.

Homogenous FRET-based fluorescent immunoassay for deoxynivalenol detection by controlling the distance of donor-acceptor couple.

Semiconductor quantum dots (QDs) are one of the most popular luminescent labels that are widely used in food and medical analysis. Their unique optical properties establish QDs as excellent tools for highly sensitive biosensors based on Förster resonance energy transfer (FRET). To provide a convenient analytical system with long-term optical stability, a FRET pair consisting of QDs as energy donor and gold nanoparticles (GNs) as energy acceptor was developed. Careful selection of donor and acceptor properties allowed to achieve a large Förster distance of 12.9 nm and to use full-size specific antibody. As the immunoreagents pair, mycotoxins were bound to proteins and then to GNs, while QDs were conjugated with specific antibodies. FRET was observed as a result of the immunocomplex formation. Contributions of FRET and inner filter effect on the quenching were evaluated separately. The quenching effect in the donor-acceptor pair was compared for proteins with different sizes. The developed homogeneous FRET-based immunoassay for the detection of deoxynivalenol (DON) is an example of a fast method for high-throughput control of mycotoxins. The quenching effect of FRET was observed with a limit of detection of 28 µg kg-1 of DON in spiked wheat samples.

Capacitive sensing of an amphetamine drug precursor in aqueous samples: Application of novel molecularly imprinted polymers for benzyl methyl ketone detection.

Highly selective molecularly imprinted polymers (MIPs) towards benzyl methyl ketone (BMK) were synthesized for application as recognition elements in a capacitive sensor. A computational approach was employed to select the most appropriate monomers and cross-linkers. Using the selected compounds, different polymerization techniques and protocols were compared in order to study the effect on the MIP performance and characteristics. MIPs synthesized by bulk polymerization using itaconic acid and 1-vinylimidazole as monomers and p-divinylbenzene as cross-linker possess the highest affinity towards the target analyte. Prior to capacitive analysis, the developed particles were immobilized on the surface of gold transducers using tyramine as a linker. The validity of the developed sensor was checked by the BMK detection in spiked tap water and real water samples. A linear working range from 50 to 1000 µM was found while the limit of detection (LOD) was determined to be 1 µM in tap water. To the best of our knowledge, both the developed MIPs towards BMK and the electrochemical sensor for its detection have not been published or marketed to date.

Recent advances in electrochemical monitoring of zearalenone in diverse matrices.

The current manuscript summarizes different electrochemical sensing systems developed within the last 5 years for the detection of zearalenone (ZEN) in diverse matrices such as food, feed, and biofluids. ZEN is one of the most prevalent non-steroidal mycotoxins that is often found in pre- and post-harvest crops. Crops contamination with ZEN and animal exposure to it via contaminated feed, is a global health and economic concern. The European Union has established various preventive programs to control ZEN contamination, and regulations on the maximum levels of ZEN in food and feed. Electrochemical (bio)sensors are a very promising alternative to sensitive but sophisticated and expensive chromatographic techniques. In the current review, recent developments towards electrochemical sensing of ZEN, sorted by type of transducer, their design, development, and approbation/validation are discussed, and the use of specialized electrochemical instrumentation is highlighted.

Novel multiplex capacitive sensor based on molecularly imprinted polymers: A promising tool for tracing specific amphetamine synthesis markers in sewage water.

The development of a sensing system for amphetamine (AMP), N-formyl amphetamine (NFA), and benzyl methyl ketone (BMK) in sewage is a strict requirement for enabling the on-site detection and tracing of the consumption of AMP, and the production and/or transportation of these target analytes. The present research is therefore devoted to the development of an on-site capacitive sensing system, based on molecularly imprinted polymers (MIPs) as recognition elements. To this end, the commercially available CapSenze capacitive sensor system was miniaturized by implementing an application-specific integrated circuit (ASIC), dedicated to the bias and read-out of the chemical sensor. MIPs towards AMP were purchased, whereas the ones towards NFA and BMK were synthesized in house. Gold transducers, consisting of six working electrodes with their corresponding reference electrodes and one common auxiliary electrode, were designed together with a flow cell to enable analyses. The applied water samples were filtered through a 20 micron filter before application in the sensors' flow cell. The limits of detection in filtered sewage water were determined to be 25 µM for NFA and BMK and 50 µM for AMP. The overall performance of the sensing system was tested by analysis of blind-coded sewage samples, provided by legal authorities. To the best of our knowledge, this is the first research presenting multiplex MIP-based detection of amphetamine synthesis markers using a capacitive sensor, miniaturized via ASIC technology. The presented technique is undoubtedly a potential solution for any analysis requiring constant reliable on-site monitoring of a substance of interest.